Many Americans suffer from the non-healing wound complications associated with diabetes. The goal of this project is to elucidate the mechanisms of action of endogenous pro-healing lipid autacoids of macrophages (PLAM), which we discovered recently, in diabetic impairments of wound healing. These PLAMs include neuroprotectins (NPDs) and maresins, PLAMs are autacoids acting on the site of their biosynthesis. Macrophages (Mf) play critical roles in wound healing. In our preliminary studies, we show that healthy murine Mf and skin after wounding generate NPD1, maresin-1, and other novel lipid mediators. In contrast, the levels of these PLAMs are diminished in comparable Mf and wounds of diabetic mice. Wound PLAM levels are inversely correlated with the levels of the oxidative stress marker F4-neuroprostane, an autooxidized derivative of DHA. Moreover, we show that hyperglycemia diminishes PLAM formation by Mf of diabetic mice. We also show that treating Mf of diabetic mice with these PLAMs rescue Mf pro-healing functions in wounds of diabetic mice. These novel findings led to our following hypotheses: 1) Diabetic complications diminish the formation of PLAMs required for normal wound healing; 2) Supplementation with these PLAMs rescues the pro-healing function of diabetes-impaired macrophages on wounds and angiogenesis. We will test our hypotheses using a wound healing model of murine gene-knockout type II diabetes, Mf depletion, vascularization assessments in vivo and in vitro, and analysis via tandem mass spectrometry (MS), including Fourier transform FTMS. The Specific Aims are: Aim 1. Test the predictions about PLAM formation and its relationship with Mf and diabetic hyperglycemia and oxidative stress during wound healing. We will study wounds and Mf using our targeted LC-UV-MS/MS based mediator-lipidomic analysis to test the prediction that: 1A. the levels of F2-isoprostanes and F4-neuroprostanes representing diabetic oxidative-stress are temporally and inversely correlated with the diminished PLAM formation in wounds of diabetic mice. 1B. simulated hyperglycemia diminishes PLAM formation by Mf. 1C. Mf depletion significantly reduces the PLAM formation in wounds of non-diabetic mice. Aim 2. Test that supplementation of neuroprotectins, maresins, or novel PLAMs rescues diabetes-impaired Mf functions that promote wound healing. We will apply diabetic mouse-produced Mf treated with these PLAMs to wounds of diabetic mice to establish their actions. Aim 3. Test that supplementation with PLAMs rescues diabetes-impaired Mf functions that promote wound-healing-required angiogenesis in hyperglycemia. We will treat diabetes-impaired Mf with PLAMs under high-glucose conditions to determine if PLAMs can rescue Mf in promoting crucial angiogenic processes of wound healing. Completion of this proposed research will contribute to revealing the molecular and cellular mechanisms for novel endogenous lipid autacoids necessary to rescue diabetes-impaired macrophage functions vital to wound healing, and provide new insight for developing novel therapeutic modalities for treating wounds in diabetics.